Purification of peptides and oligonucleotides by sample displacement chromatography process and apparatus

ABSTRACT

Sample displacement chromatography is performed at low operating pressures (less than 30 bar) and/or at high sample loading (in excess of 500 mg/cm 2  column area), with product recovery being effected in a non-gradient manner. The low operating pressures permit the use of simple and inexpensive apparatus. Non-gradient product recovery allows the desired product to be allowed in solutions with advantageously high concentration. Materials which may be purified include pharmaceuticals, pharmaceutical excipients, fine chemicals, biochemicals, X-ray contrast agents, chelating agents, peptides, proteins, oligonucleotides and vaccines. Apparatus embodiments include sample displacement chromatography apparatus comprising one or more ion exchange columns in direct combination with one or more desalting columns, and multicolumn chromatography apparatus permitting switching between a series separation mode and a parallel extraction mode.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/GB98/01109 filed Apr. 16, 1998.

This invention is concerned with chromatographic separation, moreparticularly with a new and improved method of sample displacementchromatography and applications thereof, as well as with apparatususeful in such methods.

During the last 10-15 years displacement chromatography has beensuggested as a useful alternative to liquid chromatography using elutiontechniques. In elution chromatography, components of a sample aretransported along stationary phase material, e.g. in a column, by amobile solvent phase. The various components interact at differentlevels with the stationary phase material and are therefore separatedinto bands. Displacement chromatography, on the other hands, utilises asmobile phase a displacer solution which has higher affinity for thestationary phase material than do the sample components. In the case ofcolumn chromatography the sample components are thereby displaced anddriven down the column ahead of the displacer front, competing foradsorption sites and separating into individual component bands as theyproceed.

Whereas elution chromatography normally results in substantial dilutionof the sample material, displacement chromatography permits recovery ofsample components at significantly higher concentrations and generallymakes more efficient use of the stationary phase material. Furthermore,the boundaries between individual component bands tend to beself-sharpening as a result of being driven by the sharp displacerfront; the “tailing” of bands observed in elution chromatography is thusavoided. However, displacement chromatography does suffer thedisadvantage that, to achieve optimum results, operating conditions suchas the composition, concentration and flow rate of the displacersolution must be tailored specifically to individual sample types.

Sample displacement chromatography is a self-displacement techniquewhich was first proposed by Hodges et al. [J. Chromatogr. 444 (1988),pp. 349-362] for preparative purification of peptides by reversed phaseHPLC, and which does away with the need for an extraneous displacersolution. The peptide components are applied to the column input andthemselves compete for binding sites on the stationary phase as they arewashed through the column or series of columns by an appropriatesolvent. The more strongly binding components bind first and displaceless strongly binding components to further along the column(s). Thecomponents are therefore separated according to their different degreesof hydrophobicity/hydrophilicity and thus their affinity for thestationary phase material. In a representative example a shortpre-column is used to trap impurities which are more hydrophobic thanthe desired sample component, this being retained in and saturating themain column, while the more hydrophilic impurities are further displacedand so are washed out of the main column. It is suggested that the sizeof the pre-column may be adjusted to match the amount of hydrophobicimpurities present in a particular sample, whilst the size of the maincolumn may be adjusted to ensure maximum product retention and outflowof hydrophilic impurities.

Curiously Hodges et al. subsequently use gradient elution to recover thedesired product from the main column, so that the advantageous potentialof displacement chromatography for yielding relatively highconcentration product solutions is lost. This would suggest that theinitial component separation was incomplete.

Veeraragavan et al. [J. Chromatogr. 541 (1991), pp. 207-220] reportapplication of the Hodges technique to purification of proteins usinghigh performance anion exchange chromatography columns. The apparatusused was a low pressure fast protein liquid chromatographic system andagain gradient elution was employed; this was presumably felt to benecessary in light of the observation that peak overlaps were a problemin the primary separation procedure. One and two column systems arespecifically described, the former being applicable on what are said tobe the rare occasions where the desired product is either the most orleast strongly binding component. The possibility of using amulti-column system “in which theoretically every component of theprotein sample could be fixed to a column of the proper dimensions” isnoted.

Multi-column HPLC systems for sample displacement chromatography have infact been described by Hodges, inter alia in CA-A-2059114. Arepresentative illustration shows the use of ten reversed phase HPLCcolumns or column segments connected in series for the purification ofpeptide samples; after the sample material has been loaded anddistributed/separated over the train of columns, individual columns orsegments may be eluted separately, without resort to gradient elution,the desired product component being recovered in substantially pure formfrom at least one such column or segment. Advantages of this process aresaid to be that (i) it allows ten-fold greater loading than comparablegradient elution separations; (ii) it involves minimal use of costlyHPLC solvents; (iii) it requires minimal use of fraction analyses; (iv)it avoids the need to use displacer solutions during the actualseparation; and (v) operating costs in terms of solvents, columnpackings and machine usage are much lower than typical gradientelutions.

The Hodges multi-column procedure does not appear to have been widelyadopted and has been found in practice to give products withinsufficient purity as a result of inadequate resolution of the productfrom closely related impurities. Moreover, by virtue of the need tooperate HPLC procedures at high pressure, typically 80-200 bar, theapparatus required is necessarily complicated and expensive.

The present invention is based on the finding that efficient andreproducible separation of closely related sample components may beachieved using sample displacement chromatography at low operatingpressures and recovering the desired product in a non-gradient manner.The use of low operating pressures greatly simplifies apparatusrequirements, permitting the use of simpler and less expensive pumps,taps, connectors and the like than are required for HPLC systems; theconsequential low mobile phase flow rates have been found to give riseto good separation of the sample components. By avoiding use of gradientelution the separation procedure also minimises solvent requirements andfacilitates recovery of the desired product in an advantageously highconcentration.

The procedure also makes optimum use of the stationary phasechromatographic material, since at the end of a sample displacementchromatography separation the entire length of the chromatography bedwill typically be in active use. In HPLC separations, on the other hand,only a small part of the stationary phase participates in the separationprocess at any given time. Sample displacement chromatography thereforepermits a 10- to 100-fold increase in capacity for a given stationaryphase material compared to HPLC.

According to one aspect of the present invention there is provided amethod of sample displacement chromatography which comprises (i)applying a multi-component sample to one end of a chromatography bedcomprising stationary phase material having affinity for components ofthe sample, causing components of the sample to become distributed alongthe chromatography bed by passage over the bed of a non-eluting mobilesolvent phase under an operating pressure not exceeding 30 bar, and (ii)recovering a desired component of the sample from at least a portion ofthe chromatography bed under steady state (i.e. non-gradient) processingconditions.

In general, it is preferred that the desired product should be the majorcomponent (e.g. at least 50%) of its type within the sample so that itwill give rise to adequate displacement effects in respect of lessstrongly bound impurities, although less closely related products havingsubstantially different chromatographic behaviour may be present insubstantial quantities. The method is therefore suited to thepurification of products from industrial scale syntheses, whichtypically will have been optimised to give yields well in excess of 50%;a wide range of products may be purified in this way, includingpharmaceuticals, pharmaceutical aids such as excipients, fine chemicals,biochemicals, diagnostic agents such as X-ray contrast agents, chelatingagents etc. The method is also particularly well suited to purificationof peptides and oligonucleotides prepared by solid phase syntheses,where the desired product is typically obtained in 50-80% yield and iscontaminated with a number of impurities having very closely relatedstructures.

The sample may be applied to the chromatography bed in solid or liquid(e.g. solution) form or may be applied bound to or adsorbed on anappropriate solid phase material.

A variety of stationary phase materials may be employed in the method ofthe invention. The requirement that the material has affinity forcomponents of the sample is to be interpreted as requiring that thematerial has specific sites which are capable of reversible interactionwith components of the sample. Gel filtration media, in which samplesinteract with networks rather than actual sites, are thereforeinappropriate, but systems which may be used include straight andreversed phase chromatography, ion exchange chromatography, hydrophobicinteraction chromatography and affinity chromatography. Preferredembodiments of the invention include the use of reversed phasechromatography in the purification of peptides, the use of anionexchange chromatography in the purification of peptides, proteins,oligonucleotides, phosphosugars and a wide range of syntheticnon-biomolecules containing impurities with different levels of chargefrom the desired product, and the use of affinity chromatography withphenyl boronic acid-containing chromatography beds in the purificationof products containing cis-diols.

The use of sample displacement chromatography in the purification ofoligonucleotides is itself new and constitutes a feature of the presentinvention. In many cases crude synthetic oligonucleotide productscomprise a mixture of the desired full length material with impuritieswhich are shorter fragments and therefore contain fewer chargedphosphate units. Accordingly the desired product will bind more stronglyto anion exchange materials than do the impurities, which willaccordingly be displaced whilst the desired product is retained on thechromatography bed. Such oligonucleotides may therefore be simply andeffectively purified using only a single column system.

Stationary phase materials used in separation processes according to theinvention may be in any convenient form, for example as membranes, gelsor microspheres, especially monodisperse microspheres, and will usuallybe packed into one or more columns. In some embodiments it may beadvantageous to employ extended column systems having a relativelynarrow cross-sectional area, e.g. having a length:internal diameterratio of at least 500:1, for example in excess of 750:1, preferably inexcess of 1000:1, in order to maximise interaction of thesample-components with the stationary phase material. Such use ofextended columns may simplify subsequent sampling since samples may betaken from easily defined positions within the column.

One useful column system of this type comprises a stationaryphase-containing capillary tube of plastics material, for example havingan internal diameter of 0.8 mm and an overall length of 1-2 meters.Following sample displacement chromatography in accordance with theinvention, individual sample components may be obtained by cutting outselected parts of the tube and recovering bound component therefrom.Alternatively the is entire stationary phase material may be pushed outof the tube (e.g. by application of liquid pressure following removal ofmaterial-retaining end pieces) and separated into discrete portions, thedesired product being recovered from appropriate selected portions.Systems of this type are particularly useful in small scale separationsand in preliminary investigations of chromatographic behaviour performedin order to design a larger scale system.

A series of interconnecting column segments may similarly be used, withthe desired product being recovered from one or more selected segmentsfollowing chromatography. The individual segments may, for example, bein the form of discs comprising an outer ring of a plastics materialsuch as polytetrafluoroethylene, stationary phase material being presentin the annular space within each ring, for example held betweenappropriately positioned filter membranes. A plurality of such discs maybe held together in sealing contact during sample displacementchromatography and thereafter separated to permit individual processingof one or more selected discs.

Alternatively the process may be performed using multi-columntechniques. The various columns are preferably associated withappropriate taps or valves or the like so that they may be connected inseries during the sample displacement mode separation process but may beextracted individually or simultaneously in parallel during the recoverystep.

In such a system the columns may advantageously all be connected to onedual position valve configured such that in one position of the valve amobile solvent phase may be passed through the columns in series, and inthe other position an extraction solvent, displacer solution or the likemay be fed through the columns in parallel to individual samplecollecting means associated with each column. Such valves are novel andconstitute a feature of the invention. They may, for example, comprise(i) a fixed part having inlet and outlet ports for the mobile phase,inlet and outlet ports for each of a plurality of columns and anextraction solvent/displacer solution outlet port for each of saidcolumns, and (ii) a moveable part having an extraction solvent/displacersolution inlet port and linking means, e.g. appropriate channels,conduits and/or grooves, such that in a first position the mobile phaseinlet port, column inlet and outlet ports and mobile phase outlet portare connected in series, and in a second position the extractionsolvent/displacer solution inlet port is connected to each of the columninlet ports and each of the column outlet ports is connected to theextraction solvent/ displacer solution outlet port for that column.

In another advantageous form of apparatus according to the invention theindividual columns are formed in a multicolumn block, for example an8×12 block having a height of 5-30 mm and horizontal dimensions similarto a conventional 96-well microtitre plate. Such a block may be slidablypositioned between opposing plates, said plates being perforated withholes and having channels in their block-adjoining faces such that in afirst position of the block, a mobile phase may be fed via one of theplates so as to pass through the individual columns in series. In asecond position of the block, however, an extraction solvent, displacersolution or the like may be fed via one of the plates so as to passthrough the individual columns in parallel and out through holes in theother plate. The apparatus is conveniently positioned vertically, withthe mobile phase being fed to either plate, the extraction solvent ordisplacer solution being fed to the top plate and the bottom plate beingarranged such that the individual samples from each column are fed toseparate microtitre plate wells.

It has been found that method of the invention permits the efficientseparation of sample components at substantially higher loadings ofsample per unit area of column than have hitherto been used. Hodges inCA-A-2059114 describes a peptide separation procedure using sixseries-connected 3 cm length×4.6 mm internal diameter HPLC columns andstates that optimum separation is achieved with a sample loading of 24mg, higher loads leading to some loss of the desired product. In thissystem the optimum 24 mg load corresponds to a load:area value of 145mg/cm². However, use of load:area values in excess of 500 mg/cm² e.g. ofat least 1000 mg/cm² and advantageously in the range 3000-7000 mg/cm²,has been found to result in highly efficient separations; both low andhigh pressure sample displacement chromatography techniques using suchload: area values represent a further feature of the present invention.It will be appreciated that the overall length of the column(s) will beselected to match the column capacity to the amount of sample applied,it being desirable in most embodiments of the invention that there issufficient stationary phase material to bind substantially all thesample material.

The present process in practice also uses greater sample loadings perunit column volume than does the Hodges procedure. Thus Hodges' 3 cmlength×4.6 mm internal diameter columns each have internal volumes ofca. 0.5 ml, so that application of a 24 mg optimum loading to a seriesof six columns corresponds to a load of 8 mg peptide per ml ofstationary phase. The present process, on the other hand, operatessuccessfully at loadings of 10-40 mg of samples such as peptides per mlof reversed phase stationary material and loadings of 50-100 mg ofsamples such as peptides, proteins and oligonucleotides per ml of ionexchange stationary material.

It will be appreciated that this use of high loadings to obtain highresolution separations, coupled with the high capacity of sampledisplacement chromatography separations, means that the method of theinvention makes especially efficient use of stationary phase material ina particularly simple, convenient and inexpensive manner.

The requirement that the mobile solvent phase used in separationprocesses according to the invention is non-eluting is to be interpretedas indicating that the solvent is capable of transporting less-retardedsample components to the next available binding sites but has little orno ability to interfere with interactions between the dissolved samplecomponents and the stationary phase material. A wide range of solventsystems may be used subject to this requirement. Aqueous systems usefulin, for example, reversed phase and ion exchange chromatography includewater, buffer solutions, solution of bases such as sodium hydroxide,ammonium bicarbonate or ammonium hydroxide, and solutions of acids suchas acetic acid or trifluoroacetic acid.

The versatility of separation procedures according to the invention withregard to solvent system usage has a number of beneficial effects. Inone advantageous embodiment of the invention a sample such as a peptideis obtained from a polymer support/synthesis resin, e.g. bybased-induced cleavage, applied directly to the chromatography bed, andsubjected to sample displacement chromatography using a solution of thebase or other cleavage reagent as the mobile phase. This avoids the needfor intermediate isolation of the cleavage product and permitssimplified handling of the product and the use of simplified apparatusin which the solid phase synthesis system is directly coupled to thechromatography system. The invention may also permit the purification ofproducts from solution phase synthesis system in similar manner.

In general, the mobile solvent phase will be applied until the samplehas been distributed over at least a part, preferably substantially all,of the stationary phase material; in some embodiments it may beappropriate to allow less retarded components to be washed away from thechromatography bed. Application of the mobile phase may therefore, forexample, be stopped (i) once a predetermined volume (e.g. 1-3 columnvolumes) of the mobile phase has been applied, (ii) when a particularcomponent appears at the outlet end of the chromatography bed, or (iii)when washed out material ceases to appear at the outlet end of thechromatography bed.

As noted above, in low pressure separations in accordance with theinvention, the mobile solvent phase is applied at a pressure notexceeding 30 bar, e.g. less than 15 bar, preferably less than 10 bar.Operating pressures of around 3 bar may advantageously be employed forcommercial separations; overpressures as low as 0.5 bar have been foundto give good separations. It may be appropriate to use even lower or nooverpressure, depending on the nature of the chromatography bed. Thus,for example, where the bed comprises relatively coarse material, e.g. acoarse polymer gel, the mobile solvent phase may be capable of movementas a result of gravity and/or interaction with the bed material; in suchembodiments it may be appropriate to apply a very low overpressure suchas 0.1 bar to control rather than to drive the flow of mobile solventphase.

The pressure source which drives the mobile solvent phase mayadvantageously be a pressurised gas such as nitrogen, thereby avoidingthe need for mechanical pumps and consequently reducing the operatingcosts of the process.

Recovery of the desired product may be achieved by any appropriatemethod, for example by extraction into an aqueous solvent system or anon-aqueous solvent system (e.g. an organic solvent or supercriticalcarbon dioxide), by use of a displacer solution having higher affinityfor the stationary phase material than does the sample (e.g. aqueousacetonitrile or acetic acid in the case of reversed phase chromatographyor a salt solution in the case of ion exchange chromatography), by adecrease in salt concentration (e.g. in the case of hydrophobicinteraction chromatography), or by pH change. The use of steady stateconditions inherently permits the product to be obtained in moreconcentrated form than is possible using gradient elution and since therecovery procedure is totally independent from the separation procedure,the conditions for recovery may be optimised to ensure maximumefficiency of product release and maximum efficiency of use of solvents,displacer solutions etc. without in any way compromising the efficiencyof the separation. It is accordingly possible to recover products inconcentrations 10- to 100-fold higher than those which may be obtainedwith gradient elution.

The ability to extract products into solvents of choice is alsoadvantageous in allowing recovery of products in stabilised form or inan optimum form for use in subsequent processing steps. Thus, forexample, a protein may be extracted using buffer solution at a pHappropriate to maximise stability of the protein.

In embodiments of the invention in which salt solutions are used todisplace products such as proteins or oligonucleotides, for example fromion exchange chromatographic media, the resulting product samples mayadvantageously be passed directly to individual desalting columns inorder to remove the salt as part of the extraction process. Such use ofion exchange and desalting columns in combination is itself novel andconstitutes a feature of the present invention.

An important advantage of separation processes according to theinvention is that they may be scaled up in a reproducible and consistentmanner simply by increasing the cross-sectional area of the column indirect proportion to the increase in sample loading. Thus, for example,separation of a 100 μl crude peptide sample on a 0.8 mm internaldiameter column contained a reversed phase chromatography medium andseparation of a 1.6 ml sample using a system of columns with internaldiameters of 3.2 mm (i.e. a 16-fold increase in cross-sectional area)and containing the same chromatography medium gave substantiallyidentical product profiles. Similarly, purification of thepharmaceutical excipient methoxy-polyethylene glycol phosphate using a1.6 mm internal diameter anion exchange column was found to bereproducibly scaleable up by a factor of 1000 to a column system with 5cm internal diameter. This reproducibility permits variables such as thenature and form of the stationary phase material and the nature, pH andlinear flow rate of the mobile phase to be optimised in small scaleexperiments, whereafter the sample load and column area may beproportionally scaled up to levels appropriate forpreparative/commercial separations. It should be noted in this contextthat where a constant pressure is employed to drive the mobile phase,e.g. a constant gas pressure or a mechanical pump providing a setpressure, use of such a constant pressure will give the same mobilephase linear flow rate whatever the cross-sectional area of thecolumn(s) if other parameters are unchanged.

The reproducibility and therefore the predictability of separationsaccording to the invention has the advantage that multiple separationsrun in parallel will generate consistent and predictable results; suchprocedures may therefore be used with minimum effort in the simultaneouspurification of products from multiple synthesisers, for example such asare used in combinatorial chemistry. Such purifications may readily beoperated independently of each other, for example by use of appropriatemultiway valves etc.

In general, where a sample proves particularly difficult to separate, itmay be subjected to a plurality of consecutive purification proceduresin accordance with the invention, for example using cation exchangechromatography followed by anion exchange chromatography, anion exchangechromatography followed by reversed phase chromatography, or hydrophobicinteraction chromatography followed by ion exchange chromatography. Suchtandem purifications, e.g. in which one or more bound/adsorbed samplefractions obtained by the first method are further purified by thesecond method without intermediate isolation, may readily be performedwith apparatus incorporating appropriate multiswitching valves.

The invention may advantageously be applied to situations in whichmacromolecules are modified and an improved purity profile is requiredfor the product; this may, for example, occur in vaccine production orprotein-protein conjugation. If a macromolecule shows high homogeneityaccording to a particular separation method and that method also showshigh purity for a haptene, the method may be applied to the finaldiscrimination between the various substitution levels obtained in aconjugation reaction. Representative examples of such embodimentsinclude separation by anion exchange chromatography of a proteinconjugated to a negatively charged small molecule, and separation byhydrophobic interaction chromatography of a protein-protein conjugate inwhich the two proteins differ in their chromatographic behaviour.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, which serve to illustrate the inventionwithout in any way limiting the same:

FIG. 1 is a schematic representation of a dual position valve useful incontrolling multicolumn chromatographic apparatus in accordance with theinvention;

FIG. 2 is a schematic representation of a chromatographic apparatus inthe form of a multicolumn block; and

FIG. 3 is a chromatograph showing the reproducibility of sampledisplacement chromatography separations carried out at different scales.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1 in greater detail, (a) shows a fixed valve part 1containing mobile phase inlet port 2, column inlet ports 3,3′. . . ,column outlet ports 4,4′. . . , mobile phase outlet port 5 andextraction solvent/displacer solution outlet ports 6,6′. . . FIG. 1 (b)shows moveable valve part 7 containing extraction solvent/displacersolution inlet port 8, linking means 9,9′. . . permitting serialconnection of ports 2,3,4,3′,4′. . . ,5 in one position of said part 7,linking means 10,10′. . . permitting parallel connection of extractionsolvent/displacer solution inlet port 8 and column inlet ports 3,3′. . .in the other position of said part 7, and linking means 11,11′. . .permitting connection of column outlet ports 4,4′. . . and extractionsolvent/displacer solution outlet ports 6,6′. . . in said other positionof said part 7. FIG. 1(c) illustrates serial flow of mobile phase frominlet port 2 through columns 9,9′. . . to outlet port 5. FIG. 1(d)illustrates parallel flow of extraction solvent/displacer solution fromport 8 through columns 9,9′. . . to outlet ports 6,6′. . .

In FIG. 2, (a) shows a top plate 12 and bottom plate 13 adapted tocontact an 8×12 multicolumn block assembly, with the channels used inserial separation mode shown in bold and the disconnected channels asbroken lines. As shown in FIG. 2(b), mobile phase 14 is fed viathe′bottom plate 13 and passes through the columns in series. In FIG.2(c) the channels and holes employed in parallel extraction mode areshown in bold and the disconnected channels as broken lines. As shown inFIG. 2(d) this configuration permits extraction solvent/displacersolution 15 to be fed via the top plate 12 through the columns inparallel.

The following non-limitative examples serve to illustrate the invention.Unless otherwise stated, analytical HPLC was performed using BeckmanSystem Gold apparatus equipped with a 126 gradient pump, a 168 diodearray detector, a 507 autosampler and System Gold software control.

EXAMPLE 1 Purification of Crude Peptide by Low Pressure Reversed PhaseSample Displacement Chromatography

A crude sample of the decapeptide YADKITEDLK was prepared by solid phasesynthesis using Fmoc protocol on a Biolynx 4070 automatic synthesiser,with 5- to 10-fold excesses of coupling reagents. The product was shownby HPLC analysis to have a purity of 65%.

The chromatography column comprised a 50 cm length×1.6 mm internaldiameter Teflon® tube fitted at its ends with appropriate frits andunions. The column was packed with 15 μm Resource RPC monodisperseparticles (Pharmacia), applied as a slurry in methanol and subsequentlyequilibrated with 0.1% v/v aqueous trifluoroacetic acid (hereinafter0.1% TFA).

A portion of the crude decapeptide (30 mg) was dissolved in 0.1% TFA(400 μl) and this solution was applied to the inlet end of the column.Further 0.1% TFA was applied under a gas pressure of 1.8 bar, resultingin a mobile phase flow rate of 10 μl/minute. After passage of 2 ml of0.1 TFA the gas pressure was turned off, the bottom end piece wasremoved from the column and the stationary phase material was removedand divided into 75 equal sized portions using a Multiscreen filtrationsystem (Millipore). The portions were each extracted with 10% v/vaqueous acetic acid to recover peptide sample fractions. HPLC analysisshowed that the purity of the peptide in selected fractions hadincreased to 92%.

EXAMPLE 2 Comparison of Low Pressure Reversed Phase Sample DisplacementChromatography and Conventional HPLC in Purification of Crude Peptide

The product investigated was a crude solid phase-synthesised peptidesample containing approximately 60% of the decapeptide YADKITEDLKtogether with at least three deletion sequences and a proportion ofpartially protected decapeptide.

A portion of this product (62 mg) was applied to a conventional 25 cmlength×22 mm internal diameter preparative reversed phase HPLC column(Supelcosil C-18) and gradient eluted with 0.1% TFA: acetonitrile (5-15%v/v) at a flow rate of 10 ml/minute over a period of 60 minutes;separate fractions were collected for each minute of elution. Afterfraction analysis by HPLC eight fractions were combined and lyophilisedto give 32 mg of decapeptide in 87% purity, corresponding to a yield of75%.

A further portion of the crude product (25 mg, the calculated maximumcolumn capacity) was applied to a chromatography column comprising a 2 mlength×0.8 mm internal diameter Teflon® tube packed with 15 μm ResourceRPC monodisperse particles as described in Example 1. 0.1% TFA wasthereafter applied at a flow rate of 10 μl/minute until 2 μl thereof hadbeen passed into the column, whereupon the flow was stopped. Asuccession of samples were then cut from both ends of the tube andextracted with 0.1% TFA:acetonitrile (50%); the extracts were analysedby HPLC. Once samples containing sufficiently pure decapeptide had beenidentified the remaining uncut tube was extracted in its entirety with0.1% TFA:acetonitrile (50%); the resulting solution was lyophilised togive 10 mg of decapeptide in 90% purity, corresponding to a yield of60%.

This confirms that low pressure sample displacement chromatography inaccordance with the invention permits separation of a desired peptidefrom closely-eluting impurities with comparable efficiency topreparative HPLC techniques requiring substantially more complexequipment. The process of the invention also achieves a 10- to 100-foldreduction in organic solvent requirements, yields a more concentratedproduct solution and makes maximum use of the stationary phase material.

EXAMPLE 3 Scaling up in Purification of Crude Peptide by Low PressureReversed Phase Sample Displacement Chromatography

In a small scale experiment a portion of a crude solid phase-synthesisedYADKITEDLK decapeptide sample was purified as described in Example 1except that (i) the column dimensions were 2 m length×0.8 mm internaldiameter and (ii) 10 mM aqueous sodium bicarbonate was used in place of0.1% TFA; the initial volume of the peptide solution applied to thecolumn was 100 μl. The subsequent flow of aqueous sodium bicarbonate wasmaintained at a rate of 10 μl/minute and then stopped after 200 minutes,whereafter the stationary phase material was removed and divided intoequal portions for analysis. Substantially pure product was found tooccur in the samples obtained from between 20 and 60 cm from the columninlet; these samples were pooled, extracted and analysed by HPLC.

A larger scale experiment utilised five 20 cm length×3.2 mm internaldiameter columns similarly packed with 15 μm Resource RPC monodisperseparticles and connected in series. To adjust for the 16-fold increase incolumn cross-sectional area the initial volume of peptide solutionapplied to the first column inlet was 1.6 ml; the flow rate of aqueoussodium bicarbonate was likewise increased to 160 μl/minute in order tomaintain the same linear flow rate. After 200 minutes this flow wasdiscontinued and the second and third columns (which correspond to the20-60 cm from inlet part of the tube in the small scale experiment) wereextracted together. The thus-obtained product was analysed by HPLC andfound to exhibit a substantially completely identical chromatogram tothe 20-60 cm product from the small scale experiment, as shown in FIG. 3of the accompanying drawings.

EXAMPLE 4 Purification of Bacitracin by Low Pressure Cation ExchangeSample Displacement Chromatography Combined with Reversed Phase SampleDisplacement Chromatography

Commercially available bacitracin consists mainly of bacitracin A (ca.40%) and the aminoacid-substituted analogues thereof bacitracin B₁, B₂,and B₃ (ca. 10-15% each); the balance (ca. 20%) consists predominantlyof other cyclic peptide variants and non-defined components.

Sepharose SP fast flow cation exchange material (Pharmacia) slurried in20% v/v aqueous ethanol was packed into a 21 cm length×2.4 mm internaldiameter Teflon® tube and equilibrated with 0.1% v/v aqueous acetic acid(hereinafter 0.1% AA). Bacitracin (100 mg) dissolved in 0.1% AA (1 ml)was then applied to the column, whereafter further 0.1% AA was appliedat a flow rate of 50 μl/minute for 60 minutes, the back pressure fromthe column remained constant at 0.5 bar throughout this treatment. Thestationary phase material was then removed from the column and dividedinto 24 equal portions using a Multiscreen filtration system; theportions were each extracted with pH 6.8 phosphate buffer containing 1 Msodium chloride to recover bacitracin fractions. HPLC analysis showedthat fractions from the middle part of the column had increased purity,containing ca. 60% of bacitracin A and ca. 35% of bacitracin B₁-B₃; thenature of the remaining 5% of material was not investigated.

The above procedure was repeated, except that after the separation thecolumn was cut at 6 and 14 cm from its inlet. The middle part of thecolumn was then placed in front of a 50 cm length×1.6 mm internaldiameter column similar to that described in Example 1 except that thereversed phase material therein had been equilibrated with 0.1% AA. 1 mlof 2 M sodium chloride in 0.1% AA was then applied to the resulting twocolumn system, leading to displacement of bacitracin from the firstcolumn onto the second column, where a reversed phase sampledisplacement chromatographic separation took place. Division of thesecond column and extraction of appropriate parts thereof permittedrecovery of salt-free product comprising ca. 75% of bacitracin A, ca.20% of bacitracin B₁-B₃ and less than 5% of impurities.

EXAMPLE 5 Purification of Bovine Serum Albumin by Low Pressure AnionExchange Sample Displacement Chromatography

Eight 20 cm length×3.2 mm internal diameter columns were filled with QSepharose fast flow anion exchange material (Pharmacia) slurried in 20%v/v aqueous ethanol; the material was then equilibrated with pH 6.8phosphate buffer. The columns were connected via two Omnifit 1164 valvessuch that they could be operated in series or in parallel.

With the columns connected in series, a solution of bovine serum albumin(Sigma A 2153—1.2 g) in pH 6.8 phosphate buffer (10 ml) was applied tothe first column, whereafter further buffer was applied at a flow rateof 0.4 ml/minute for 100 minutes. The flow was then discontinued and thevalves were switched so that each column was treated in parallel with pH6.8 buffer containing 1 M sodium chloride at a flow rate of 0.5ml/minute; the outflow from each column was passed through a Hitrapdesalting column and collected in 1 ml fractions. In this way salt-freepurified protein fractions were obtained at concentrations in excess of100 mg/ml buffer; the total volume of buffer/solvent consumed during theprocess was less than 200 ml.

EXAMPLE 6 Isolation of Protein-Haptene Conjugates with SpecificSubstitution Levels

A protein material exhibiting homogeneous migration in anion exchangechromatography, for example as obtained in Example 5, was treated withthe net negatively charged compound diethylene-triamine pentaacetic acid(activated by reaction of its triethylammonium salt with a subequimolaramount of diisopropylcarbodiimide in dimethylformamide). The agent wasadded to the protein solution in portions, and the course of thereaction was monitored by analytical anion exchange chromatography,which permitted identification of the various substitution levels.

Once a major portion of the protein was shown to be monosubstituted, theproduct mixture was separated by anion exchange sample displacementchromatography as described above, using a single 50 cm length×0.16 mminternal diameter column filled with the Q Sepharose material. Thestationary phase material was then removed and divided into eight equalportions, which were extracted with 1 M sodium chloride solution andanalysed.

Depending on the processing conditions and the particular chemistry usedfor modification, some 10-100 mg of homogeneous substituted protein maybe obtained from this final separation. The simplicity of operation ofseparation procedures according to the invention, the high resolutionachievable, the low solvent volume requirements and consequent highconcentrations obtainable in respect of end product solutions, and thepossibility of combining steps such as ion exchange chromatography anddesalting, result in it being possible to carry out the entire procedurein a single day.

EXAMPLE 7 Purification of Oligonucleotide by Low Pressure Anion ExchangeSample Displacement Chromatography

A solution of 25 mg of a crude solid phase-synthesised thiolated 21-meroligonucleotide in concentrated aqueous ammonium hydroxide (the reagentused to cleave the oligonucleotide from the synthesis resin) was appliedto the inlet end of a chromatography column comprising a 2 m length×0.8mm internal diameter (i.e. 1 ml volume) Teflon® tube packed with 15 μm QSepharose fast flow anion exchange material equilibrated withconcentrated ammonium hydroxide. Further concentrated ammonium hydroxidewas then applied at a flow rate of 5 μl/minute for 400 minutes.Thereafter the flow was stopped, the outlet end filter was removed andthe stationary phase material was pushed out and divided into twelveequal portions which were placed in filtering cups, water-washed andextracted with 10 mM sodium hydroxide solution containing 2 M sodiumchloride to yield oligonucleotide sample fractions. The samples weredesalted using NAP-10 columns (Pharmacia) and selected samples wereanalysed by capillary gel electrophoresis. Samples from the firstquarter of the column contained 93-97% pure product, whereas samplesfrom the second half were free of oligonucleotide material. Deletionsequences were found in samples from the intervening quarter of thecolumn. Accordingly, since only about half of the stationary phasematerial was used in binding oligonucleotide material, a total of about50 mg of the crude oligonucleotide per ml column volume may be separatedusing this system. The results indicated that at least 50% of thedesired product could be recovered in 95% purity (as determined bycapillary gel electrophoresis).

EXAMPLE 8 Purification of Methoxy-polyethylene Glycol Phosphate by LowPressure Anion Exchange Sample Displacement Chromatography

Methoxy-polyethylene glycol phosphate (MPP) is used as an excipient inpharmaceutical formulations and must therefore be prepared with a purityin excess of 98%. Impurities present in MPP as synthesised may includepolyethylene glycol diphosphate, methoxy-polyethylene glycol, MPPmonophenyl ester and MPP diphenyl ester. It is therefore desirable toperform a purification step after synthesis.

90% pure MPP (50 g) was dissolved in water (1 l) and the pH was adjustedto 8.8 by titration with concentrated ammonia. A sample of this solution(1 ml) was applied under a 3 bar pressure of nitrogen gas to a 1 mlength×1.6 mm internal diameter Teflon® tube packed with Q Sepharose FFin the acetate form, and was followed by water (4 ml) under a similar 3bar pressure. Sampling of the column contents (by GPC analysis with aTSK G3000SWXL column and using 5 mM phosphate. (pH 7) as the mobilephase) showed that the first 5 cm contained the stronger bindingpolyethylene glycol diphosphate along with MPP, whereas the last 20 cmcontained MPP monophenyl ester along with MPP; the uncharged MPPdiphenyl ester molecules were not retained and so passed straightthrough the column. The MPP content between 5 and 80 cm was extractedusing 0.5 M hydrochloric acid and lyophilised to give dry MPP (38 mg).GPC and NMR analysis showed an impurity content of less than 1%.

This trial separation was then scaled up 1000-fold, using four 5 cminternal diameter columns packed with acetate form Q Sepharose FF to bedheights of 5, 25, 25 and 25 cm (total column volume 1.6 l), and applying1 l of sample under a 3 bar pressure of nitrogen gas. A further 2 l ofwater were applied to distribute the sample along the length of thecolumn system; the effluent from the column was found to contain thesame components as the 80-100 cm portion of the column in thesmall-scale trial separation taken together with the material displacedfrom the column. Extraction of the three 25 cm columns as describedabove yielded MPP (37 g) in a solution volume of 1.3 l. GPC and NMRanalysis showed a purity in excess of 99%.

EXAMPLE 9 Purification of N,N′-dipyridoxylethylenediamine-N′N-diacetateby Low Pressure Anion Exchange and Cation Exchange Sample DisplacementChromatography

The chelating agent N,N′-dipyridoxylethylenediamine-N,N′-diacetate(PLED) is normally synthesised by alkylation of the correspondingdiamine, e.g. using bromoacetic acid. Since the diamine contains atleast four potential alkylation sites the synthetic product willinevitably possess a somewhat complex impurity profile, so that it isdesirable to perform a purification step after synthesis.

Crude PLED was prepared by dissolving N,N′-dipyridoxylethylenediamine inaqueous sodium hydroxide at a concentration of ca. 25 g/l and adjustingthe pH to 11. Aqueous bromoacetic acid (70% of the stoichiometricamount) was added to the solution and the pH was adjusted to 11.1. Theresulting mixture was heated to 50° C. and the reaction was monitored bycapillary electrophoresis (75 μm fused silica, 50 mM borate, pH 9.2,containing 1 mM diethylenetriamine pentaacetic acid). The reactiongenerated a mixture of monoalkylated, dialkylated and trialkylatedproducts, in the ratio 1:2:1 as determined by UV absorbance at 214 nm.The yield of PLED was approximately 30%.

The reaction mixture was applied without work up to a column packed withQ Sepharose FF and separated using water as the mobile solvent phase(0.5 bar nitrogen pressure), thereby separating the components accordingto their substitution levels and leading to removal of lower chargedimpurities, which were displaced from the column. Following thisseparation, the column contained approximately 10 g/l material with anestimated product purity of 45% and quantitative recovery. The columnwas extracted using 1M acetic acid and applied directly to an SPSepharose FF column at a loading of 50 mg/ml. Separation was effectedusing 1 M acetic acid as the mobile solvent phase (4 bar nitrogenpressure). Trialkylated impurities were displaced, whereas thedialkylated PLED product was recovered from the last part of the columnin approximately 50% yield and with a purity of 93%. The first part ofthe column contained material that had not been displaced in the firstpurification step; some 25% of contaminated PLED product was found inthe intermediate part of the column.

EXAMPLE 10 Purification of Iodixanol by Low Pressure Sample DisplacementBorate Affinity Chromatography

The X-ray contrast agent iodixanol is a dimer of two tri-iodinatedaromatic rings. It contains four vicinal diol functions and thereforemay be purified by affinity chromatography using resins containingphenyl boronic acid functions which coordinate cis-diols.

A crude sample of iodixanol (<80% purity) was dissolved in 0.1 M aqueoussodium carbonate and applied to a column packed with the phenyl boronicacid function-containing polyacrylamide gel Affi-Gel 601 (Biorad), at aloading of ca. 100 mg sample/mL gel. Further solvent was applied under apressure of 0.1 bar nitrogen, at a flow rate of approximately 50μl/minute. Flow was stopped after 90 minutes and the column was dividedinto six parts which were extracted using aqueous acetic acid. Analysisby reversed phase HPLC (Shimadzu LC-8, Brownlee C-18 column, 5-17%acetonitrile gradient over 20 minutes) confirmed the almost completedisplacement of impurities and the isolation of product with over 93%purity and a yield in excess of 50%.

EXAMPLE 11 Purification of a Phosphosugar by Low Pressure Anion ExchangeSample Displacement Chromatography

Vaccine structures based on chemical sequences from bacterial cell wallsare synthesised as repeating units comprising carbohydratephosphodiesters and, optionally, linking groups. Such a structure may bepurified by applying 2 mg of a sample to a 20 cm length×0.5 mm internaldiameter column packed with 15 μm Source Q anion exchange material(Pharmacia). After separation the column is divided into 1 cm portionswhich are extracted with 2 M aqueous sodium chloride and analysed by GPC(TSK G3000SWXL, 5 mM phosphate, RI detection). Earlier parts of thecolumn show product with high purity, whilst additional peaks relatingto shorter chain length compounds are observed in later parts of thecolumn.

The purified product may be used for conjugation to macromolecules toobtain immune responses to hemofilus influenza virus B.

EXAMPLE 12 Purification of an Oligonucleotide Using an Anion ExchangeColumn Coupled to a Desalting Column

Crude solid phase-synthesised oligonucleotide is obtained inconcentrated aqueous ammonium hydroxide, the reagent used to cleave theoligonucleotide from the synthesis resin. The cleavage solution isapplied directly at a loading of 50-100 mg/ml to an anion exchange resinequilibrated in concentrated ammonium hydroxide, and is separated usingfurther concentrated ammonium hydroxide as the mobile solvent phase.This results in displacement of less charged impurities such as shorterfailure sequences, leaving the desired oligonucleotide on the resin at apurity in excess of 95%. The resin is washed with water and extractedwith 2 M aqueous sodium chloride. The saline extract is passed directlyto a desalting column to separate the extracting salt from theoligonucleotide product; the latter product is thereby obtained inpurified and highly concentrated form (50-100 mg/ml).

What is claimed is:
 1. A method of sample displacement chromatographywhich comprises (i) applying a multi-component sample to one end of achromatography bed comprising stationary phase material having affinityfor components of the sample, causing components of the sample to becomedistributed along the chromatography bed by passage over the bed of anon-eluting mobile solvent phase under an operating pressure notexceeding 30 bar, and (ii) recovering a desired component of the samplefrom at least a portion of the chromatography bed under steady stateprocessing conditions.
 2. A method as claimed in claim 1 wherein thestationary phase material comprises straight phase, reversed phase, ionexchange, hydrophobic interaction or affinity chromatographic material.3. A method as claimed in claim 2 wherein the stationary phase materialcomprises reversed phase chromatographic material and the samplecomprises one or more peptides.
 4. A method as claimed in claim 2wherein the stationary phase material comprises anion exchangechromatographic material and the sample comprises one or more peptides,proteins or oligonucleotides.
 5. A method as claimed in claim 1 whereinthe stationary phase material is in the form of monodispersemicrospheres.
 6. A method as claimed in claim 1 wherein the mobilesolvent phase is applied under an operating pressure not exceeding 15bar.
 7. A method as claimed in claim 6 wherein the mobile solvent phaseis applied under an operating pressure not exceeding 10 bar.
 8. A methodas claimed in claim 7 wherein the mobile solvent phase is applied underan operating pressure of about 3 bar.
 9. A method as claimed in claim 1wherein the pressure applied to the mobile solvent phase derives from apressurised gas.
 10. A method as claimed in claim 1 wherein thestationary phase material is packed within one or more columns and thesample is applied in an amount exceeding 500 mg per square centimeter ofthe internal cross-sectional area of said column or columns.
 11. Amethod as claimed in claim 10 wherein the sample is applied in an amountof at least 1000 mg per square centimeter of the internalcross-sectional area of said column or columns.
 12. A method as claimedin claim 11 wherein the sample is applied in an amount of 3000-7000 mgper square centimeter of the internal cross-sectional area of saidcolumn or columns.
 13. A method as claimed in claim 1 wherein thestationary phase material is packed within one or more columns such thatthe ratio of overall column length to column internal diameter is atleast 500:1.
 14. A method as claimed in claim 13 wherein said ratio isin excess of 750:1.
 15. A method as claimed in claim 14 wherein saidratio is in excess of 1000:1.
 16. A method as claimed in claim 1 whereinthe desired component is recovered by application of a salt solution toat least a portion of the chromatography bed and the thus-obtainedproduct is passed through a desalting column.